Recognize that enzymes are key components of chemical reactions in all living things.

Understand and apply the scientific method.

Practice safe laboratory procedure.

Unit Objectives:

Upon completion of this unit, the student will be able to:

Differentiate between protease and amylase enzymes in terms of function and target molecules.

Understand the role of enzymes during the germination of common seed plants.

Interpret quantitative and qualitative results.

Gather and analyze data from various enzymatic reactions and use this data to make conclusions.

Explain how temperature, concentration, and pH affect the activity of enzymes.

Background:

Living things
utilize a simple sugar called glucose to provide energy for life
processes. Organisms link excess glucose molecules together to form the
molecule called starch for food storage. The organism cannot directly
utilize starch molecules when it needs energy. It must first break down
starch into glucose. This process requires the help of protein
molecules called enzymes. Enzymes help break the bonds in a starch
molecule so that the organism can use the smaller glucose units for
energy.

Each type of
enzyme controls specific types of reactions. Amylases are enzymes that
target starch molecules. Human saliva contains an amylase. Another
group of enzymes works primarily on protein molecules. These enzymes
are known as proteases. Proteases degrade proteins into smaller pieces
known as amino acids. Proteases can be found in common products such as
meat tenderizer, pineapple, and papaya.

This series of
activities is designed to explore the concept of an enzyme. The concept
is developed through a series of investigations that manipulate
specific variables. Students draw their own conclusions about enzyme
activity by comparing and analyzing both quantitative and qualitative
data.

This unit is
designed using an inquiry-based approach to learning science. The
students should be led and guided to ideas and concepts by the teacher.
The unit is designed as a series of activities, not necessarily a day
by day unit. In an inquiry-based environment, students are given time
to explore answers to their own questions, further influencing their
comprehension of the content. The students are asked to design their
own experiments. A logbook or journal is recommended. A suggested way
of performing the experiment is provided with each activity. Use this
only as a guideline.

Advance Teacher Preparation:

You will need the
following materials to complete 3 important tasks before the unit
begins. The following materials are based on a class size of 30
students.

Plastic petri dishes (science catalog)

6/group (lids can be used)

Viable wheat seeds

30/group of three(approximately 15 grams)

Viable corn seeds

30/group of three(approximately 100 grams)

Viable barley seeds

30/group of three(approximately 15 grams)

Paper towels

3/group of three

Distilled water

8 liters

Agar (dry powder or tablets)

22 grams

Tincture of iodine (2% iodine)

30 mL

Knox Brand unflavored gelatin

5 boxes (28 grams each)(4 envelopes per box)

Corn Starch

1 box

Meat tenderizer (unspiced)

100 gram bottle

Papaya Enzyme(available in tablet form from stores such as GNC — check labels for papain)

approx. 90 tablets/bottle

Bromelain Enzyme (optional)

approx. 90 tablets/bottle

Amylase Enzymes(available in tablet form from stores such as GNC — check for amylase)

approx. 90 tablets/bottle

Mortar and pestle (small)

1

Hot plate

1

Stirring rods

2

400 mL beakers

2

Preparing the dishes:The teacher should
prepare a total of 12 dishes for each group of students. 6 dishes
should be made with gelatin and 6 dishes should be made with agar.
Suggested mix proportions:

Agar: 2 grams of powdered agar per 200 mL of water
Gelatin: 2 envelopes of gelatin per 200 mL of water

Before heating, add to the mixture 420-mg of starch per 200 mL water for the agar mixture
only. Heat until boiling and pour into dishes. The dishes do not need
to be covered — use the lids to double the quantity of available
dishes. The agar will set quickly. The gelatin will need a little more
time to completely set. These may be prepared 24-48 hours before they
are needed. Dishes may be covered with wax paper after they cool.

Preparing the enzyme solutions:Several enzyme
solutions will need to be made. For the first activity, you will need
two "unknown" solutions. For this, use one amylase and one protease. We
suggest preparing the solutions as follows:

Amylase solution: a small amount of saliva with 5 mL of water.
Protease solution: 2 grams of meat tenderizer in 5 mL of water.
Stir both until well mixed or dissolved.

Germinating the seeds:Begin this process at least five days prior to the beginning of the unit.
Use C-fold paper towels if possible. Take approximately 30 seeds of the
same type and place them in the fold of the paper towel. Roll the paper
towel with the seeds inside and moisten with distilled water. Label the
package as Day 1 / seed type (corn, barley, or wheat). Repeat this
process with the other types of seeds. Enclose all three types from day
one in aluminum foil and label the outside. There should be sets
exactly like this one prepared for each group of three students.

Repeat this
process each day for at least four more days in order to provide each
group with five sets of seeds on the first day of the unit.

Safety Notes:

Iodine wash is mildly irritating to the skin and eyes. Safety glasses
are recommended when using iodine solutions. Iodine solutions will
permanently stain clothing, books, notebooks, and other porous
materials. Iodine solutions will also stain skin but it will wash off
within a few days.

Food coloring
is not toxic. However, ingestion of anything in a science lab is not
recommended. Food coloring will permanently stain clothing and other
porous surfaces. Stains on skin will wash off within a few days.

Activity 1: What are Enzymes and How Do They Change Living Things?

Objectives:

To use seeds in different stages of germination in developing the concept of an enzyme.

To interpret
experimental data in determining the presence or absence of starch in
food sources and in different types of biological media.

small samples of different foods such as apple, potato, bread, ham, flour, sugar, and cheese for each group

dry wheat, barley, and corn seeds for each group

Provide each
group with five sets of seeds. Each set of seeds will contain seeds
that began germination on a different day. In each set, there will be
seeds from corn, barley, and wheat.

Ask the students to come up with a list of ways in which the seeds are different depending on which stage they are looking at.

Discuss the
idea that some parts of the seed disappear over the course of 5 days.
Come up with ideas on where these parts "go". Also discuss where the
"new" parts come from. Lead them to the idea that the plant needs food
to grow, just like people do. Where does the plants' food come from?

Provide each
group of students with a gelatin plate and an agar-starch plate. Each
group will also be given two unknown solutions (A & B). They should
put a drop of each unknown on each of the plates and label them. It is
recommended that the plates be allowed to sit for about 24 hours.
Ensure that students label their plates. It is not necessary to cover
plates.

To introduce
the concept of starch, have several food items available for each
group. Show the class what iodine looks like out of the bottle
(brown-yellow in color). Have the students place a few drops of iodine
solution on a small piece of bread. They will see that the iodine turns
black. Have them do the same with other food items. A few
recommendations are ham, cheeses, apple, potato, flour, and sugar.

Now, tie this
concept into the plant seeds. Take a dry corn, wheat, and barley seed
and put iodine on each of them. The students should begin to conclude
that foods like bread and seeds are made up of the same thing because
they react the same way to an iodine test. With teacher facilitation,
they should realize that the substance that reacts with iodine is
starch, a stored carbohydrate that is used as a food reserve in plants.
The plant embryo inside of the seed uses this starch for energy to grow.

Distribute the
dishes from step 4 (the A/B dishes) to the students (these dishes
should have been sitting for 24 hours). Both of the dishes will be
translucent but colorless in appearance. Allow the students to add an
iodine wash solution to both plates. Swirl the solution around the
plates and leave them sit for a few minutes. Pour off any additional
iodine into the sink drains.

Ask the students the following leading questions and have them record their responses:
a. Do you see any changes in the dish?
b. What is the substance in the dish?
c. What are the differences in the two dishes?

Allow the
groups time to come to these conclusions. The students should conclude
that there was starch in the agar plate and that the iodine turned the
starch blue/black. They should also note what they see in the gelatin
plate. They will explore that shortly.

What happened
in the clear areas of the agar plate? Students should conclude that
whatever was in the unknown solutions accounted for the difference.
Explain to them that these "unknown" solutions contain proteins that
break down substances into smaller particles. In this case, the starch
was broken down, resulting in an area that did not stain.

Have the
students work as a group to determine why the plates responded
differently. They should come to a point where they conclude that the
plates are made out of two different substances and that the unknown
solutions acted differently depending on what the plates' material was.

Introduce the
concept of an enzyme. Explain that enzymes are very specific for the
substances that they work on. Explain that agar is a vehicle for
suspending starch. Amylases are enzymes that break down starch. On the
other hand, gelatin is a protein. Proteases are enzymes that break down
proteins. Some examples of substances containing enzymes are saliva,
pineapple, papaya, LACTAID, and meat tenderizer.

Review the
results from the two A/B dishes. Explain that where the amylase was
working, there was a clear area when stained with iodine. Where a
protease was working, the area appeared to be "watery" (in breaking
down the protein, a molecule of water is formed when two amino acids of
the protein are separated).

Activity 2: Dilution Process

Objective:

To learn how to prepare a serial dilution from a concentrated substance.

Suggested Time Period:

One 45 minute period

Materials:

Food coloring

six small (1 oz) condiment cups

distilled water

eye droppers or pipettes

overhead projector

transparency sheet (use a new sheet)

Frequently, the
need for solutions of different concentrations arises. In order to set
up solutions of identical chemical composition with varied
concentrations, it is easiest to use a serial dilution process. A
serial dilution process starts with a solution that is designated as a
"concentrate". Using the concentrate, successively weaker solutions are
mixed. This technique is handy for use in a case where the experimenter
wishes to analyze the effect of varying solution concentrations.

Mark the condiment cups as follows:

1:1

1:10

1:100

1:50

1:500

1:1000

The cup for each dilution step will have a total of ten drops in it, just before proceeding to the next dilution step.

Place ten drops
of food coloring into the cup marked 1:1. This means that there is 1
drop of concentrate for 1 drop of total liquid volume in the cup.

Use the pipette
to place one drop from the 1:1 cup into the cup marked 1:10. Rinse the
pipette with distilled water. Add nine drops of distilled water to the
cup. There is now 1 drop of concentrate for 10 drops of total liquid in
the cup, also known as a 1:10 dilution.

Use the pipette
to place one drop from the 1:10 cup into the cup marked 1:100. Rinse
the pipette with distilled water. Add nine drops of distilled water to
the cup. There is now 1 drop of 1:10 for 10 drops of total liquid in
the cup, also known as a 1:100 dilution.

Use the pipette
to place one drop from the 1:100 cup into the cup marked 1:1000. Rinse
the pipette with distilled water. Add nine drops of distilled water to
the cup. There is now 1 drop of 1:100 for 10 drops of total liquid in
the cup, also known as a 1:1000 dilution.

Use the pipette
to place 2 drops from the 1:10 cup into the cup marked 1:50. Rinse the
pipette with distilled water. Add 8 drops of distilled water to the
cup. There are now 2 drops of 1:10 for 10 drops of total liquid in the
cup, also known as a 2:100 dilution, which can be mathematically
reduced to 1:50.

Use the pipette
to place 2 drops from the 1:100 cup into the cup marked 1:500. Rinse
the pipette with distilled water. Add 8 drops of distilled water to the
cup. There are now 2 drops of 1:100 for 10 drops of total liquid in the
cup, also known as a 2:1000 dilution, which can be mathematically
reduced to 1:500.

As solutions
become more diluted, it is successively more difficult to detect any
color in the solutions. Label a new transparency sheet as follows:

1:1

1:10

1:100

1:50

1:500

1:1000

H20

Place the
transparency sheet on the overhead projector and turn on the lamp.
(Make sure it is level) Place one drop of each solution (use H20
for your control) on the transparency sheet near each corresponding
dilution, and allow the heat from the lamp to evaporate the water from
each drop. Even the drops that seem to have no color in them will show
up as a small dot of coloring.

Other dilution levels may be achieved by remembering to divide the drops of "concentrate" by the total volume of liquid.

Activity 3: Examine How Concentration Affects Enzyme Activity

Objective:

To determine if the concentration of enzyme in a solution affects its activity.

Suggested time period:

Two or three 45 minute class periods

Materials:

meat tenderizer

amylase tablets

distilled water

test tubes, test tube racks, and pipettes for dilution

stirring rods

mortar and pestle

balance and weighing boats (an index card, folded to form a V-shape works)

agar and gelatin plates

marking pen

iodine solution

graph paper and ruler (optional)

Students will
naturally think that if a little enzyme works, then more enzyme must
work better! Give them the opportunity to test this hypothesis. They
should be working with meat tenderizer (papain) for the protease and a
commercial amylase tablet for their amylase.

They should begin this part of the process after completing the dilution exercise in Activity 2.

Allow the
students to come up with a procedure to test how concentration of the
enzyme affects the digestion of starch or protein. Their experimental
design may differ from one group to the next. Encourage all feasible
options. The best method for us was to take 5 g of Adolph's Meat
Tenderizer and add it to 5 mL of distilled water, stirring until
dissolved. This will give you a "concentrated" protease solution. This
solution should be the starting point for their dilution series. To
make the amylase solutions, use one tablet dissolved in 5 mL of
distilled water to begin. Make it more concentrated by adding
additional tablets to the same volume of water. Concentrations from 1-5
tablets should be sufficient. Allow the tablets some time to dissolve
on their own. The students can then use either a stirring rod or mortar
and pestle to help dissolve the tablets.

The students
should label their dishes with the type of medium and the solutions
they used on the bottom of the dish. Allow the dishes to sit for
approximately 24 hours.

Examine the
students' plates the next day for amylase activity (as evidenced by
clear areas after an iodine stain) and protease activity (hydrolyzed
area of the medium).

Students should
conclude that a more concentrated enzyme shows a greater amount of
activity, shown by a wider area. If their results appear to show a
trend, you may wish to have them graph their data with concentration on
the x-axis and size of area in mm on the y-axis.

Activity 4: How Does Length of Germination Affect Enzyme Activity?

Objective:

To examine seeds for an increase or decrease in enzyme activity over time.

Suggested Time Period:

Two 45 minute class periods

Materials:

agar plates (3 per group)

one corn seed from each day of germination plus a dry seed (6 total)

one barley seed from each day of germination plus a dry seed (6 total)

one wheat seed from each day of germination plus a dry seed (6 total)

scalpel or exacto knife

marking pen

Remind students
that plant seeds contain starch as the primary food reserve. Some plant
seeds store protein as the primary food reserve, and others store
lipids (fats and oils) as the primary food reserve. Have students
propose a hypothesis to answer this question: What happens to the stored starch in a germinating seed as days pass?
Growing plants use amylase enzymes to break down starch so that glucose
may be utilized for energy. Proteins are broken down by proteases, and
lipids are broken down by lipases.

The students
will test their hypothesis using the above materials in an experimental
design that you help them develop. They should observe that the starch
is stored inside of a seed coat. Therefore, if you really want to know
how much starch is still present and how actively the enzyme is
degrading the starch, the seed will have to be opened. The best way
that we found to do this is by taking the seeds at each day of
development and splitting them with a sharp knife. Supervise your
students carefully while doing this or you may want to split the seeds
for them.

We achieved the
best results when we removed the embryo and took half of a seed,
placing it cut-side down on the agar surface. We placed one half-seed
for each day of germination on one dish. Keep each type of side on one
dish.

You will have
to allow time for this assay to work. When sufficient time has passed
(24 hours), allow the students to stain the plates with iodine
solution. The largest clear area should be around the "oldest" seeds.
Have the students make conclusions as to why this happened and to
compare the results to their original hypothesis. Also, it may be
interesting to have them compare the appearance of the iodine stain in
all three types of seeds.

Activity 5: Extracts from Germinating Seeds

Objective:

To analyze the enzyme activity level in liquid extracts from seeds at different stages of germination.

Suggested Time Period:

Two - three 45 minute class periods

Materials:

3 agar-starch petri dishes per group

12 small (1 oz) condiment cups

Distilled water

Eye dropper or pipette

Iodine wash

1 Mortar/pestle per group

5 seeds of each (wheat, barley, and corn) germinating for 5 days

5 seeds of each (wheat, barley, and corn) germinating for 3 days

5 seeds of each (wheat, barley, and corn) germinating for 1 day

5 seeds of each (wheat, barley, and corn) which are still dry

Germinating
seeds produce an amylase to digest food stored in the seed as a
polysaccharide known as starch. The starch is broken down into the
monosaccharide called glucose, which can be used directly by the
growing plant embryo.
Q. Is the level of enzyme the same or is it different for seeds in various stages of germination?
Q. How can we design an experiment to answer this question?
Q. What can serve as a control for the experiment?

One suggested
experiment design: (if class time does not allow for all three types of
seeds, use one or two types of seeds — crushing the seeds in the mortar
will take about three to four minutes for each type and each
germination stage)

Label petri
dishes, one for each type of seed extract, and put five marker spots on
the underside of each petri dish. Label the spots: H2O, dry, 1 day, 3 day, and 5 day.

Crush five
seeds with 20 drops of water using the mortar and pestle (follow the
same procedure for both germinating seeds and dry seeds). Pour the
resulting slurry into a small plastic condiment cup and label it. (e.g.
Wheat — 5 day extract) Rinse the mortar and pestle with distilled water.

Repeat the procedure for each of the different number of days of germination and for each type of seed.

Place one drop
of each wheat solution (try to draw only the liquid into the pipette)
on the wheat petri dish. Repeat this procedure for the barley and the
corn petri dishes. Place a drop of distilled water on the H2O spot.

Extracts may be discarded or saved for advanced activity (See #9)

Set petri dishes in a safe place to sit overnight. Covers may be used, but they are not required.

After about
twenty-four hours, visually examine petri dished for any change. Record
observations. Why are some locations different in appearance?

Stain the petri
dish with a weak iodine wash. After about two minutes, drain the iodine
wash from the petri dishes and rinse gently with distilled water from a
squeeze bottle. Examine the petri dishes after staining. Record
observations and have group members discuss the possible reasons for
any differences in appearance between each location on a given petri
dish.

Advanced activity: This activity requires at least 12 glass culture tubes (5mm x 70mm is a good choice, but any very small test tubes will suffice)

Q: Does heating or boiling an enzyme extract render it unable to digest starch?

A suggested experiment design to test this: Place the liquid from each
extract in a separate glass culture tube and immerse the bottom of each
tube in a boiling water bath for about sixty seconds or until it just
starts to boil (be careful not to boil the samples dry — some samples
will have very little liquid to work with). Use a long, thin pipette to
draw a drop of boiled extract. Place the drop on a fresh set of
agar-starch petri dishes (as in step 6 above). Allow the petri dishes
to stand for about 24 hours, then stain with iodine wash. Compare
results with the results from the live enzyme extracts.

Activity 6: How Does pH Affect the Activity of an Enzyme?

Objective:

To change the pH of enzyme solutions with common products to determine how enzyme activity is affected

The students
should be starting to understand the specificity of enzymes. One factor
that influences enzyme activity is pH. Depending on your students'
background, you may have to introduce this concept first. pH level is a
measure of the amount of particular types of ions produced by a
compound when it is put in solution. A substance that is an acid
releases H+ ions when placed in solution. Substances that are acidic have pH values below 7.0. Strong acids have a lower pH and release H+ ions almost completely in solution. Other substances release OH-
ions in solution. These solutions are called bases and have a pH above
7. Stronger bases have a higher pH and almost completely dissociate
into OH- ions. pH of 7.0 is neutral.

Tell the
students that a common acid is vinegar (acetic acid) and a common base
is baking soda (sodium bicarbonate). Have a volunteer from each group
"donate" some saliva into two weighing boats.

Label two
agar/starch petri dishes. One dish will be used to test the effect of
acids on saliva (which they have already found has amylase in it) and
the other will be used to test the effects of bases. Solicit ideas from
the students on how they might do this. They may need some assistance
if the concept of pH is new.

They can use
the vinegar as is out of the container, since it is already a diluted
form. Make a solution of baking soda by mixing approximately 700 mg of
powder with about 10 mL of water.

Test the pH of
saliva before starting by having the student volunteer put a pH test
strip on their tongue. After they add the vinegar and baking soda to
separate samples, take the pH of the saliva mixture and compare it to
the original. Be sure to take the pH of the vinegar and baking soda
without saliva also. The students should record all data from these
test strips so that they can see how pH changes with the addition of an
acid or base.

Now the
students should test to see how this pH change affects the ability of
amylase to digest starch. We suggest that the students have three test
areas on each plate. One area will be saliva only. One area will be
saliva mixed with either vinegar or baking soda, and the third area
will be a drop of the vinegar or baking soda solution only.

Allow the
dishes to sit about 24 hours. The students should then stain them with
iodine solution to see where starch is still present. They should make
observations about their results in their logbooks.